Identidad de la sustancia y conexión con el modelo corpuscular

Cortical slice preparation: Mice were anesthetized with pentobarbital sodium and decapitated after disappearance of corneal reflexes. Brains were rapidly

removed and cut at 350 µm using a vibrating microtome (Leica VT1000S or VT1200S) in ice-cold dissection buffer containing (in mM), 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 75 sucrose, 10 D-(+)-glucose, 1.3 ascorbic acid, 7 MgCl2, and 0.5 CaCl2, bubbled with 95% O2 and 5% CO2. Slices recovered for 20 min at 35°C in ACSF containing: (in mM) 124 NaCl, 3 KCl, 1.25 Na2PO4, 26 NaHCO3, 1 MgCl2, 2 CaCl2, and 20 D-(+)-glucose, saturated with 95% O2, 5% CO2 and kept at room temperature for at least 40 min. Recordings were made in a submersion chamber at 30-32°C in the same ACSF except in low Mg2+ _{conditions where 1} mM MgCl2 was excluded or when drugs were added as noted. Ifenprodil, MK- 801, and picrotoxin were purchased from Sigma (St. Louis, MO). All other pharmacological agents were purchased from Ascent Scientific (Weston-Super- Mare, UK).

Whole-cell recordings: L2/3 pyramidal cells were visually identified with IR-DIC optics and pyramidal morphology was confirmed in the majority of neurons by filling them with Alexa 488 (0.01% wt/vol). Patch pipettes were pulled from thick-

walled borosilicate glass with open tip resistances of 2-7 MΩ when filled with internal solution containing: (in mM) 20 KCl, 100 (K)Gluconate, 10 HEPES, 4 (Mg)ATP, 0.3 (Na)GTP, 10 (Na)Phosphocreatine with pH adjusted to 7.25 and osmolarity adjusted to ~295 mOsm with sucrose. In all experiments, the internal solution contained the NMDAR antagonist MK-801 (1 mM) to block postsynaptic NMDARs, including GluN3A-containing (Das et al., 1998), and to ensure isolation of AMPAR-mediated responses (Berretta & Jones, 1996; Corlew et al., 2007; Rodriguez-Moreno & Paulsen, 2008). Cells were recorded in either voltage- or current-clamp configuration with a patch clamp amplifier (Multiclamp 700A; Molecular Devices), and data were acquired and analyzed using pCLAMP 9.2 or 10 software (Molecular Devices). Series and input resistances were monitored throughout experiments by measuring the response to a -5 mV step at the beginning of each sweep. Series resistance was calculated using the capacitive transient at the onset of the step and input resistance was calculated from the steady-state current during the step. No series resistance compensation was applied.

mEPSC recordings: Similar to previously described (Corlew et al., 2007), AMPA receptor-mediated mEPSCs were recorded in the presence of tetrodotoxin (200 nM) and picrotoxin (50 µM) at negative holding potentials (-80 mV) to facilitate block of postsynaptic NMDARs. Minimal glycine (1 µM) was also added to bind the preNMDAR co-agonist binding site without saturating postsynaptic NMDARs (Li & Han, 2007). We measured mEPSC amplitude and frequency during a

baseline period at the beginning of the experiment and during bath application of an NMDAR antagonist. D-AP5 (50 µM) and ifenprodil (3 μM) were bath applied for 10 minutes and all other pharmacological agents were applied for 15 minutes. Events with a rapid (<3 ms) rise time and exponential decay were identified using an automatic detection template. Quantification of mEPSCs was calculated from the percentage change in frequency or amplitude of the last 5 minutes of

NMDAR antagonist application normalized to the last 5 minutes of baseline. Cells were only included for analysis if (1) there was < 30% change in Rinput and Rseries, (2) there was < 100 pA change for Iholding, and (3) Rseries was < 30 M.

Evoked glutamate release: Recording solutions for these experiments and tLTD experiments were the same as in mEPSC recordings except for the omission of tetrodotoxin, picrotoxin, and glycine from the ACSF. For all evoked and tLTD experiments, L2/3 pyramidal cells were recorded in voltage- or current-clamp while L4 axons were stimulated extracellularly every 15 seconds with a two- conductor cluster electrode with 75 µm tip separation (FHC Inc., Bowdoin, ME). To analyze the effect of UBP141 on non-NMDAR currents, the effect of 15 minute application of UBP141 on single AMPAR EPSCs was determined in high divalent ACSF (4mM Ca2+ _{and 4mM Mg}2+_{) containing 50 µM picrotoxin and 100} µM D,L-AP5.

For current-clamp experiments measuring evoked glutamate release or tLTD, current was injected to maintain a -70 mV resting potential if necessary; cells were excluded from analysis if input resistance changed more than 30% or

if >200 pA of current was required to maintain a -70 mV resting potential. To measure the effect of D-AP5 on evoked glutamate release, the amplitude and paired pulse ratio (PPR; second EPSP/first EPSP) of two EPSPs evoked at 30Hz were measured. After a 10 minute baseline during which monophasic and fixed latency EPSPs maintained no change in slope or amplitude, D-AP5 (50 µM) was applied for 10 minutes. Change in the amplitudes of the first and second EPSPs was quantified as a percentage of baseline (the last 5 minutes of D-AP5 / the last 5 minutes of baseline). In a single instance, D-AP5 application in a wildtype animal reduced both EPSPs to noise levels and was excluded from analysis because it was not possible to properly assess release probability via PPR.

tLTD induction:To demonstrate that tLTD in wildtype mice was homosynaptic, EPSPs were alternately generated by one of two bipolar stimulating electrodes placed in L4 and L2/3 of V1 except in tLTD experiments in GluN2D–/– _{mice and in} a subset of experiments comparing tLTD in wildtype and GluN3A–/– _{mice. A} steady baseline was recorded for 10 minutes during which monophasic and fixed latency response EPSPs were maintained with no change in amplitude or slope. The tLTD induction period consisted of 100 action potentials (APs) at 0.2 Hz each followed within 15-20 ms by an EPSP generated selectively by L4

stimulation. Postsynaptic APs were produced by a brief (< 5 ms) depolarization of the postsynaptic L2/3 cell and EPSPs generated in L4 were produced in an identical manner as the baseline period. Change in EPSP slope was calculated

as the percentage decrease in slope from the last 10 minutes post-LTD normalized to the last 5 minutes of baseline.